rna self-assembly · 2006-03-30 · 3/29/06 labean compsci 296.5 ellington & szostak nature....

3/29/06 LaBean COMPSCI 296.5

RNA Self-assembly

(2004) Science 306, 2068-2072


RNA Self-assembly(2004) Science 306, 2068-2072


RNA Self-assembly (2004) Science 306, 2068-2072


stemmer

2003

Rational designDirectedevolution


RNA Physical Catalyst

(2004) Science 304, 850-852

*UTP is 5-(4-pyridylmethyl)-uridine 5’ triphosphatePd2(DBA)3 is dibenzylide-acetone palladium (0)


*UTP is 5-(4-pyridylmethyl)-uridine 5’ triphosphatePd2(DBA)3 is dibenzylide-acetone palladium (0)

RNA Physical Catalyst


Ellington & Szostak

Nature. 1990 Aug 30;346(6287):818-22.

In vitro selection of RNA molecules that bind specificligands.

Ellington AD, Szostak JW.Department of Molecular Biology, Massachusetts General Hospital, Boston 02114.

Subpopulations of RNA molecules that bind specifically to a variety oforganic dyes have been isolated from a population of random sequenceRNA molecules. Roughly one in 1010 random sequence RNA moleculesfolds in such a way as to create a specific binding site for small ligands.


Ellington & Szostak Nature. 1990 Aug 30;346(6287):818-22.

Random DNA

Random RNA

Transcription

Column fractionation

Unbound (discard)[flow-through high salt]Bound RNA

[high salt wash, water elute]

T7 Promoter cDNA Priming

ReverseTranscription Dye column



100 random nucleotides (+ constant regions) Complexity of initial pool ~1015 (1060 possible) Poor transcription; PCR amp; ~1013 diversity ~4x1014 RNA molecules applied to column <0.1% of initial RNA library bound then after 4 to 5

cycles of bind, RT, PCR, Transcribe, rebind ~50% ofRNA molecules bound.

Binding is specific for dye selected. For 1 column 20 clones were sequenced ~16-20

nucleotides were invariant over the set of clones. APTAMER from Latin ‘aptus’, to fit.



AC A C G C A C U C A GAAAA GUAC A C C UCACA A-C CC A UCC AUCC CCAAC CAG GC CGG GCC A A ||| |||| ||||| ||| || ||| ||| U G AGG UAGG GGUUG GUC-CG GCC CGG U C A-UAA ACCC C-GAC C-A-A AAU CA G-C G-C C | G-C C-G C-G C-G U-A U-A A \ A G G-C A-U G-U G G

B4 Reactive blue binder


Tuerk & Gold

SELEX- systematic evolution of ligands byexponential enrichment.

Selected RNA for binding to T4 DNA polymerase. 65,536 possible sequences. Wild-type T4 bacteriophage mRNA sequence found

and a variant with 50% identity to wild-type.


Tuerk & Gold


Bartel & Szostak

220 random positions 1.6x1015 diversity


Bartel & Szostak


In vitro evolution of nucleic acids.

Random pool

Select for desired phenotype

Selected poolMultiplecycles

Final selected ensemble

Amplify and/or mutate geneotype


Review

In vitro selection requires linking of genotype andphenotype.

Binding targets: simple ions, small molecules,peptides, proteins, organelles, and cells.


Review AMP binding aptamer

Selectivity: theophyllineaptamer shows 10,000-folddecrease in binding tocaffeine which differs byone additional methyl group.


Review


In vitro selection and directedevolution of peptides and proteins

Goal: find protein/peptide with desired function(binding, catalyst, stability, etc.)

In vivo vs In vitro selection. Rational design vs. Library methods. Combinatorial peptide libraries.

Geysen. Discontinuous epitope mapping. Solid phasepeptide synthesis. Birth of combinatorial chemistry.

Phage display. mRNA fusion. Ribosome stalling. Antibody engineering and evolution. Primerless PCR. DNA shuffling. Sexual PCR.


Scott & Smith

Filamentous bacteriophage (M13, fd)

Cloning into p3 (pIII) coat protein

NNK codons: N=ACTG, K=GT.Encodes 32 triples, all 20 aminoacids and only one stop codon.

antigenantibody

epitope

antigen combining region


Scott & Smith

64 million possible hexapeptides Library contained 1.3 x 1014 phage from

2 x 108 clones


Phage display

Enzyme selection. Note: hidden in vivo

selection too...


mRNA-proteinfusion

Ribosomedisplay

Other. Cro?


Phage display of Ab libraries


Artificial immune maturation

Gene library from human,animal, or synthetic source.

Maturation: Saturation mutagenesis of

CDRs. Chain shuffling. Growth in mutagenic E. coli

strains.


Diabodies and triabodies

In vitro selection/evolution of bifunctional antibodyfragments.

Universal protein-based adapter/address system.


Stemmer

http://www.maxygen.com/science-pub.php

Gene reassembly Whole plasmid reassembly


Stemmer

Can natural diversity accelerate the evolution process? Goal: obtain moxalactamase activity from four

cephalosporinase genes. DNA shuffling within genesor between genes...


Stemmer


Stemmer

Can natural diversity accelerate the evolution process? Goal: obtain moxalactamase activity from four cephalosporinase

genes. DNA shuffling within genes or between genes... One cycle: ~8-fold improvements from separately evolved genes. One cycle: 270- to 540-fold improvement from the four genes

shuffled together. Best clone contained 8 segments from 3 of the 4 genes & 33

amino-acid point mutations.


stemmer

2003


Belcher(2000) Nature 405, 665-668


Belcher

GaAs (100),GaAs(111), InP,GaN, ZnS, CdS,Fe3O4, CaCO3,etc.

(2000) Nature 405, 665-668

rna self-assembly · 2006-03-30 · 3/29/06 labean compsci 296.5 ellington & szostak nature....

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